Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

When activated by Ca2+CaM, MLCK phosphorylates the SMM RLC at Ser19, which activates the actin-SMM ATPase leading to the cyclic interactions of SMM with actin required for smooth muscle contraction.1-3 In smooth muscle, MLCK is in low abundance relative to SMM with a ~1:20 molar ratio of MLCK to SMM and an even lower molar ratio of activated MLCK to SMM.4-11 Yet upon agonist activation of smooth muscle, Ca2+CaM-MLCK phosphorylates as much as 60% of the SMM in a relatively short (second) time.12-15 Clearly, determining the kinetics of i) SMM Ca2+CaM-MLCK activated phosphorylation, ii) MLCK dissociation from pSMM, iii) and selective rebinding of MLCK to SMM is central to understanding the rate-limiting mechanisms of smooth muscle activation, and the factors that may influence smooth muscle contractility. MLCK is tightly associated with the contractile apparatus.16-18 It contains both myosin and actin binding sites and has been shown to bind both actin and myosin in solution. The C-terminal telokin domain of MLCK binds to the SMM heavy chain at the junction between the two head domains and the tail domain. This places the MLCK catalytic core close to the two RLC subunits bound to each head domain. The stoichiometry of binding is one MLCK per molecule of SMM.19 Little is known about the kinetics of SMM phosphorylation by Ca2+CaM-MLCK. Sellers and coworkers have shown that SMM phosphorylation significantly decreases the MLCK-SMM affinity in the absence of ATP (Kd from 0.8 μM to >100 μM).20 However, it is unclear whether SMM phosphorylation decreases the MLCK-SMM association rate or increases the MLCK-SMM dissociation rate. The specific kinetic steps (association, phosphorylation, or dissociation) that limit the SMM phosphorylation rate have not been identified. Since spectroscopic signals of association and dissociation that would be useful for standard transient kinetic studies in solution have not been developed, we have taken a non-spectroscopic approach. We have developed an in vitro model system in which smooth muscle proteins are attached to a coverslip surface in various configurations. This approach allows us to: (i) control the constituents of the system, (ii) measure the kinetics of SMM phosphorylation by MLCK and subsequent development of actin motion driven by surface-attached pSMM, and (iii) directly observe the interactions of single MLCK molecules with single SMM molecules under similar conditions using TIRF microscopy. We previously characterized MLCK and CaM-MLCK complexes that co-purify with SMM from chicken gizzard muscle.18 With the CaM-MLCK-SMM complexes attached to a coverslip surface with SMM in the monomeric form, we showed that the MLCK was activated by Ca2+ to phosphorylate SMM, initiating actin filament sliding in an in vitro motility assay. Half-maximal actin-sliding velocity was observed at pCa50 6.1, similar to other in vitro and in vivo studies, suggesting that the CaM-MLCK was activated by Ca2+ in a physiological manner. We also showed that the MLCK reversibly interacted with the surface-attached SMM through its telokin domain, which is the known SMM-binding site on MLCK. Here we use this in vitro system to correlate the rate of MLCK-induced SMM activation with the association and dissociation kinetics of single MLCK molecules interacting with surface-attached monomeric SMM (not filamentous). We found that the SMM phosphorylation rate of 1.17 s−1MLCK−1 is similar to the rate at which actin motility is activated and is comparable to the SMM-MLCK dissociation rate constant of 0.80 s−1 obtained from our single molecule experiments. We also show that MLCK dissociates from SMM and pSMM at essentially the same rate, implying that the decrease in SMM-MLCK affinity upon SMM phosphorylation 20 results from a decrease in the pSMM-MLCK association rate. This suggests that in the MLCK-SMM kinetic cycle, dissociation of MLCK from pSMM is rate-limiting and that the rate of the intrinsic phosphorylation step is faster than this dissociation step. Using our measured dissociation rates and Kds, and estimates of SMM concentrations in muscle, our measurements suggest that association to unphosphorylated SMM (11-46 s−1) would be much faster than to pSMM (0.1-0.2 s−1) in the muscle environment.